The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells’ ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. ‘Rounding’ of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with
Toxoplasma gondii
or
Salmonella typhimurium
. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.
When transmitted through the oral route,
Toxoplasma gondii
first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The
in vitro
culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling
T. gondii
infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of,
T. gondii
. Using quantitative label-free mass spectrometry, we show that
T. gondii
infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that
de novo
synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between
T. gondii
and the host intestinal epithelium influence the course of infection.
Single cell RNA sequencing (scRNA-seq) is a powerful tool in detailing the cellular landscape within complex tissues. Large-scale single cell transcriptomics provide both opportunities and challenges for identifying rare cells playing crucial roles in development and disease. Here, we develop GapClust, a light-weight algorithm to detect rare cell types from ultra-large scRNA-seq datasets with state-of-the-art speed and memory efficiency. Benchmarking on diverse experimental datasets demonstrates the superior performance of GapClust compared to other recently proposed methods. When applying our algorithm to an intestine and 68 k PBMC datasets, GapClust identifies the tuft cells and a previously unrecognised subtype of monocyte, respectively.
Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant and species‐specific models. Increasingly, 3D stem cell‐derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review, we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.